immuno blot

Frederick Garbrecht fgarbrec at POST.ITS.MCW.EDU
Wed Feb 10 19:54:10 EST 1993


Are your antigens denatured on the western blot (did you get them there by
blotting after denaturing electrophoresis?), and if so, do you know
whether your antibody binds to denatured epitopes?  This is a common
reason for failure of antibody binding in western blotting.  If this is
the problem, there's nothing you can do short of getting better
antibodies, unless you want to try re-naturing your antigens on the blot.
Good luck,

F Garbrecht
Med Coll Wisc
fgarbrec at post.its.mcw.edu

On 10 Feb 1993, David E Bell wrote:

> Date: 10 Feb 93 15:16:15 GMT
> From: David E Bell <bell at mari.acc-admin.stolaf.edu>
> To: methods-and-reagents at net.bio.net
> Subject: immuno blot
> 
> 
> 
> a simple experiment, but not working...
> who can help ???
> 
> I did immunoblots (running ag on a gel, blotted it, incubated blot with
> 		   positive human serum, developed with anti human POD
> 		   conjugate  ==> no bands !!)
> Ponceau staining revealed lots of protein on my blot.
> My substrate reaction is positive, too.
> After several investigations the problem is bordered in:
> 
> 	no binding of the antibodies (or not enough) in the positive
> 	patient serum to the on NC membrane presented antigens !!
> 
> why ?? did anybody experience  similar problems ??
> 
> in addition it must be said, that i did the same procedure as ELISA (same
> antigens coated, same human serum, same POD conjugate !!) - and there it was
> working very well !!!
> 
> So any ideas what prevents ab binding to NC presented antigens ????
> 
> I appreciate every hint, thougt or idea. Thanks.
> 
> Tom Pasternak
> Immunology University Aberdeen
> Internet: ugn4501 at aberdeen.ac.uk
> 
> or as sender: bell at stolaf.edu
> 





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